! Project Summary Every year, more than 100 Americans a day die after overdosing on opiates. Addiction to opioids, including prescription drug such as oxycodone, and illicit drugs such a s heroin and fentanyl, is a national crisis that affects public health and the economy. Thus, there is an urgent need to develop better treatments for opiate addiction, which requires a better understanding of its biological basis. The primary goal of our proposal is to identify cell types and cell type-specific gene expression patterns associated with higher vulnerability to compulsive oxycodone use in an unbiased and quantitative way. We will accomplish this goal by implementing single-cell sequencing assays to measure gene expression and chromatin accessibility in thousands of individual cells in a single experiment. We will use brains of N/NIH heterogeneous stock (HS) rats that have undergone the extended access to oxycodone self-administration procedure. We focus on HS rats because they are genetically diverse and exhibit an exceptional behavioral repertoire. These rats are characterized as vulnerable or resistant to oxycodone compulsive intake based on advanced analysis of addiction-like behavioral traits, including tolerance, dependence, motivation, and compulsive drug intake. This behavioral paradigm recapitulates many of the key neuroadaptations observed in human addiction and has high face, predictive, and construct validity for oxycodone use disorders. This project takes advantage of a brain tissue repository of HS rats that have been genotyped and characterized as vulnerable and resistant to compulsive oxycodone use. The oxycodone biobank (www.OxycodoneBioBank.org) will provide the samples to be used in this project. We will focus on the nucleus accumbens, a brain region involved in the transition from moderate to excessive drug use. Our preliminary studies on single-cell analysis of the cerebral cortex provide a compelling strategy to study the biological basis of opiate addiction. We propose: 1) to use single-cell RNA-seq to identify gene expression changes in brains of HS rats that are characterized as prone or resistant to oxycodone compulsive use (Specific Aim 1); 2) to use single-cell ATAC-seq to identify changes in chromatin accessibility and transcription factors binding sites in the same population of HS rats (Specific Aim 2); 3) to use H3K27Ac- PLAC-seq to link distal regulatory elements to target genes involved in oxycodone addiction-related behaviors (Specific Aim 3). This project will benefit from multiple expertise and will leverage existing resources, including those provided by the oxycodone biobank (U01DA044451) and the NIDA center for GWAS in outbred rats (P50DA037844). We believe that the proposed studies have the potential to lead to groundbreaking discoveries in the mechanistic bases of opioids addiction. The integrative analysis of our sequencing datasets will provide considerable new insights concerning the contribution of distinct cell-types to molecular changes associated with addiction related phenotypes. ! !